Nuclear hormone receptors (NRs) mediate essential physiological processes, but are also implicated in human disease. Though NR activity is highly regulated, nearly all existing approaches to treat NR-dependent disease target only one regulatory mechanism, ligand binding. Ligand-based therapies are limited by acquired resistance and side-effects associated with the alteration of normal NR activity in non-diseased tissue. My long term goal is to develop novel, non-ligand treatments for NR-dependent diseases that overcome these limitations. My current work focuses on the androgen receptor (AR), a canonical NR that is involved in many diseases, including prostate cancer (PCa). Current anti-androgen treatments inhibit AR activity in all AR-expressing tissues, resulting in serious side-effects, and development of resistance after 3-5 years of treatment. Orthogonal approaches, those that seek to inhibit AR activity by means other than ligand binding, have the potential to overcome these limitations and offer an important therapeutic complement. Combinatorial treatment with ligand-based and ligand-independent inhibitors could delay or prevent resistance. Additionally, creating tissue-selective, non-ligand inhibitors could reduce side-effects associated with current anti-androgen treatments. I have created a cell-based assay that measures the ligand-induced conformation change of AR using fluorescence resonance energy transfer (FRET). I used this assay to identify novel, non-ligand molecules that inhibited AR conformation change and subsequent transcriptional activity. The most promising compound, pyrvinium pamoate, is an FDA-approved drug that I have shown has potent anti-androgenic effects in mice and possible cell-selective activity. In this grant, I propose to determine the mechanistic basis of cell-selective PP action and correlate these findings with tissue-selectivity in vivo. I also aim to greatly expand our understanding of the cellular control of AR activity using the conformation change assay to dissect the cellular networks that regulate tissue-selective AR conformation change, thus identifying clinically relevant drug targets and new leads for anti-androgen development.